EP0116587A4 - Production of secondary metabolites from micro organisms. - Google Patents
Production of secondary metabolites from micro organisms.Info
- Publication number
- EP0116587A4 EP0116587A4 EP19830902637 EP83902637A EP0116587A4 EP 0116587 A4 EP0116587 A4 EP 0116587A4 EP 19830902637 EP19830902637 EP 19830902637 EP 83902637 A EP83902637 A EP 83902637A EP 0116587 A4 EP0116587 A4 EP 0116587A4
- Authority
- EP
- European Patent Office
- Prior art keywords
- production
- secondary metabolite
- nutrients
- fermentation
- tylosin
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P1/00—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes
Definitions
- the present invention relates to a method for increasing or prolonging the production of a secondary metabolite by a microorganism in a batch culture.
- Microorganisms including both prokaryotic and eu aryotic microorganisms grow in nutrient solutions until the nutrients required for the growth of the organism, i.e. the production of bio ass, have been consumed. This is generally termed the trophophase.
- the idiophase in which there is relatively little biomass production but in which the existing cells survive and metabolise.
- a number of microorganisms produce secondary metabolites during such an idiophase.
- secondary metabolites are generally compounds not essential for normal growth of the organism and are only produced when the organism is in a stationary phase or when there is some restriction on the metabolic pathways associated with unrestricted growth.
- secondary metabolites are the antibiotics, pharmacologically active compounds, and toxins.
- the present inventors have found that further substantial improvements in the long term rate of secondary metabolite production can be achieved by the periodic addition to the culture medium of those nutrients required for secondary metabolite production.
- the present invention consists in a method for increasing the production of a secondary metabolite by a microorganism, comprising the step of cultivating a suitable microorganism in a culture medium containing those nutrients essential for the production of the desired secondary metabolite, and adding additional amounts of those nutrients essential for the production of the desired metabolites at discrete intervals spaced apart in time.
- the present invention further consists in secondary metabolites produced by the foregoing method. While the full reason for the advantage gained by the periodic feeding of secondary metabolite precursors has not been fully elucidated it is thought that the following explanation may be correct. This explanation is given to assist in an understanding of the invention and is not to be taken as limiting the scope of the present invention. It has been found that during the trophophase there is a repression of secondary metabolite synthesis and that during the idiophase the rate of secondary metabolite synthesis peaks and then falls off. It has generally been assumed that this fall off in rate of production of the secondary metabolite is due to the depletion of the precursors for the secondary metabolites.
- the method according to the present invention is applicable to any microorganism capable of producing secondary metabolites.
- Such microorganisms include filamentous fungi, the filamentous prokaryotes such as the streptomyces and certain of the unicellular bacteria. . Many such organisms are listed in the article "Pharmacologically Active Agents from Microbial Sources" by Dr. S.L.
- the present invention is particularly applicable to the production of antibiotic secondary metabolites and more particularly to the production of macrolide antibiotics.
- the macrolide antibiotics are a structurally related group of antibiotics produced by species of Streptomyces and which all contain a large lactone ring containing 12 to 22 atoms and having few double bonds and no nitrogen atoms. These antibiotics generally have one or more sugar residues attached to the lactone ring.
- the method may also be adapted to the production of secondary metabolites by microorganisms growing on complex media as it is not uncommon for complex media to be used in the trophophase of secondary metabolite production the additional defined nutrients required for secondary metabolite synthesis can be fed during the idiophase. If the requirement of the organism for these supplemental feeds have been determined then the present invention can be used to generate cyclical feeds and stimulate production of the secondary metabolite.
- the present invention can be used to feed a carbon source, such as glucose, and an amino acid, or another source of nitrogen, in cyclical fashion assuming the lipid source is always present in excess.
- a carbon source such as glucose, and an amino acid, or another source of nitrogen
- the method may be carried out in a strictly batch process in which the nutrients essential for the secondary metabolite production are introduced into the culture medium in a concentrated form and without removal of any of the culture medium. Alternatively a proportion of the culture medium may be removed at the time of addition of the nutrients.
- the amount of the cyclical nutrient addition should be adjusted such that there is not a residual amount of that nutrient present in the culture medium during the period between the cyclical feeding of the nutrients. It will also be recognised that not all required nutrients need be fed cyclically, however, it is preferred that all those nutrients which generate catabolite repression be fed cyclically.
- Figure 2 shows the parameters of Figure 1 and glucose uptake for a cyclic fed-batch fermentation according to this invention fed at intervals of 24 hours.
- Figure 3 shows the parameters of Figure 2 for a cyclic fed-batch fermentation according to this invention fed at intervals of 36 hours.
- Figure 4 shows the effect of varying the amplitude of feeding in a cyclic fed batch fermentaiton according to this invention
- Figure 5 shows the effect of varying the period of feeding in a cyclic fed-batch fermentation according to this invention.
- the organisms used in this study was Streptomyces fradiae NRRL 2702. The conditions of growth and analytical procedures were described in Biotech, Bioeng., 22, 1785, (1980) .
- the medium used contained in one " litre of ' distilled water; Sodium chloride, lg; magnesium sulphate, 2.5g; cobalt chloride, 0.0005g; zinc sulphate, 0.0005g; ferric ammonium citrate, 1.5g; betaine hydrochloride, 2.5g; L-sodium glutamate, 17.5g; dipotassium hydrogen phosphate, 1.15g.
- the normal fed-batch fermentation was carried out by continuously feeding the culture with glutamate and glucose solutions.
- glutamate and glucose were fed at predetermined and constant time intervals in the different fermentation runs. The time intervals between these additions varied from 12 to 48 hours.
- the glutamate and glucose were fed using a Watson-Marlow pump (Model 501) and a syringe pump (Sage Instruments, Model 352). Additional methyloleate was added to maintain the concentration above 5 g/1 at all times during the fermentation.
- the feed cycles were controlled by an Apple microcomputer.
- RESULTS The pattern of the batch fermentation of tylosin is shown in Fig. 1 and Table 1 (Control Batch column) .
- the rate of tylosin synthesis was maximal between 24 and 48 hours of fermentation when the culture was actively growing.
- the glucose and sodium glutamate were rapidly metabolised by 48 hours and methyloleate was utilised as soon as glucose and glutamate were exhausted.
- the q - . was as high as 1.2 mq/g/h during growth phase but decreased with time to 0.05 mg/g/h by the end of the fermentation.
- sodium glutamate and glucose were linearly fed from 48 hours until the end of the fermentation.
- the feed rates of sodium glutamate and glucose were 0.8 ml/h—(0.18 g/ml of culture fluid) and 0.4 ml/h (0.025 g/ml of culture fluid) respectively.
- the results of this experiment were shown in Table 1 (Linear Feed column) though the kinetic pattern of the fed-batch fermentation was similar to that of the normal batch, the productivity was improved and the speci ic rate of tylosin synthesis reached the value of 0.28 mg/g/h by the end of the ferementation, which is about six-fold higher than the specific rate of the normal batch. Further improvement was obtained by cyclic fed-batch fermentation in which sodium glutamate and glucose were cyclic fed at various cycles from 48 hours to the end of the fermentation.
- Table 1 Values of observed in batch, normal and cyclic fed-batch fermentations.
- Figure 3 shows the cell dry weight, glucose concentration, glutamate concentration and tylosin concentration of the fermentation described above but fed with glucose and glutamate on a 36 hour cycle.
- glucose was fed into the culture medium for 0.25 hour at the rate of 250 mg/l/hr each 36 hours and the monosodium glutamate was fed for 0.5 hour at the rate of 1800 mg/l/hr each 36 hours .
- Tylosin concentration was measured as total tylosin by chloroform extractions as described "" in Antimicrobiol Agents and Chemotheraphy Apr. 1980 p.p. 519-525. This extract contained, in addition to tylosin, certain other macrolide antibiotics such as relomycin. The pure tylosin was recovered by high pressure liquid chromatography.
- Figure 4 shows the effect of varying the amplitude of the cyclic feeding of glucose and monosodium glutamate on the total tylosin production over a 10 day period in the above described fermentation. It will be seen that as the amount of glucose rises or falls from 0.04 g/l per 24 hours and as the amount of monosodium glutamate rises or falls from 0.6 g/l per 24 hours so the tylosin concentration rises or falls.
- Figure 5 shows the effect of varying the period of the cyclic feeding on the total tylosin concentration over a 10 day period in the above described fermentation.
- Monosodium glutamate was fed at the rate of 0.9 g/l per cycle and glucose was fed at the rate of 0.065 g/l per cycle. It can be seen that a cycle of 36 hours produces a maximum tylosin production of 2.1 g/l. This is to be compared with a normal batch feremtation which produces 0.9 g/l tylosin and a linear feed situation in which 0.9 g/l monosodium glutamate and 0.065 g/1 glucose were fed to the culture medium for a 24 hour period which produced 1.2 g/1 tylosin.
Abstract
Description
Claims
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
AT83902637T ATE58396T1 (en) | 1982-08-23 | 1983-08-23 | PRODUCTION OF SECONDARY METABOLITES FROM MICROORGANISMS. |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
AU5513/82 | 1982-08-23 | ||
AUPF551382 | 1982-08-23 |
Publications (3)
Publication Number | Publication Date |
---|---|
EP0116587A1 EP0116587A1 (en) | 1984-08-29 |
EP0116587A4 true EP0116587A4 (en) | 1986-11-27 |
EP0116587B1 EP0116587B1 (en) | 1990-11-14 |
Family
ID=3769710
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
EP19830902637 Expired - Lifetime EP0116587B1 (en) | 1982-08-23 | 1983-08-23 | Production of secondary metabolites from micro organisms |
Country Status (4)
Country | Link |
---|---|
EP (1) | EP0116587B1 (en) |
JP (1) | JPS59501772A (en) |
DE (1) | DE3381997D1 (en) |
WO (1) | WO1984000777A1 (en) |
Families Citing this family (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
GB8516415D0 (en) * | 1985-06-28 | 1985-07-31 | Celltech Ltd | Culture of animal cells |
US5686273A (en) * | 1991-08-05 | 1997-11-11 | Cultor Food Science, Inc. | Fermentation process for producing natamycin with additional carbon and nitrogen |
ES2093272T3 (en) * | 1991-08-05 | 1996-12-16 | Bio Tech Resources | CONTINUOUS PRODUCTION OF NATAMYCIN. |
GB9906206D0 (en) * | 1999-03-17 | 1999-05-12 | Biodiversity Ltd | Fermentation apparatus |
GB0818453D0 (en) | 2008-10-08 | 2008-11-12 | Novartis Ag | Fermentation processes for cultivating streptococci and purification processes for obtaining cps therefrom |
Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CH334468A (en) * | 1955-05-10 | 1958-11-30 | Ciba Geigy | Process for the production of a new antibiotic |
BE689124A (en) * | 1966-10-31 | 1967-03-31 | ||
DE1442223A1 (en) * | 1964-01-15 | 1969-11-06 | Courtaulds Ltd | Method and device for the production of a chemical substance by fermentation |
FR2320349A1 (en) * | 1975-08-06 | 1977-03-04 | Agronomique Inst Nat Rech | ENZYMATIC PROCESS USING INCLUDED MICROORGANISMS |
Family Cites Families (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US3039932A (en) * | 1958-01-07 | 1962-06-19 | Research Corp | Tissue culturing with arginine, citrulline or aspartic acids supplements to the media |
-
1983
- 1983-08-23 EP EP19830902637 patent/EP0116587B1/en not_active Expired - Lifetime
- 1983-08-23 WO PCT/AU1983/000113 patent/WO1984000777A1/en active IP Right Grant
- 1983-08-23 JP JP50274583A patent/JPS59501772A/en active Pending
- 1983-08-23 DE DE8383902637T patent/DE3381997D1/en not_active Expired - Fee Related
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CH334468A (en) * | 1955-05-10 | 1958-11-30 | Ciba Geigy | Process for the production of a new antibiotic |
DE1442223A1 (en) * | 1964-01-15 | 1969-11-06 | Courtaulds Ltd | Method and device for the production of a chemical substance by fermentation |
BE689124A (en) * | 1966-10-31 | 1967-03-31 | ||
FR2320349A1 (en) * | 1975-08-06 | 1977-03-04 | Agronomique Inst Nat Rech | ENZYMATIC PROCESS USING INCLUDED MICROORGANISMS |
Non-Patent Citations (4)
Title |
---|
CHEMICAL ABSTRACTS, vol. 95, 1981, page 459, abstract 95397h, Columbus, Ohio, US; S. BHUWAPATHANAPUN et al.: "Production of the macrolide antibiotic tylosin in fed-batch culture", & J. FERMENT. TECHNOL. 1981, 59(3), 235-7 * |
CHEMICAL ABSTRACTS, vol. 96, 1982, page 583, abstract 215986u, Columbus, Ohio, US; K. VU-TRONG et al.: "Continuous-culture studies on th. regulation of tylosin biosynthesis", & BIOTECHNOL. BIOENG. 1982, 24(5), 1093-103 * |
CHEMICAL ABSTRACTS, vol. 98, 1983, page 481, abstract 70265n, Columbus, Ohio, US; K. VU-TRONG et al.: "Stimulation of tylosin productivity resulting from cyclic feeding profiles in fed batch cultures", & BIOTECHNOL. LETT. 1982, 4(11), 725-8 * |
See also references of WO8400777A1 * |
Also Published As
Publication number | Publication date |
---|---|
WO1984000777A1 (en) | 1984-03-01 |
EP0116587A1 (en) | 1984-08-29 |
EP0116587B1 (en) | 1990-11-14 |
DE3381997D1 (en) | 1990-12-20 |
JPS59501772A (en) | 1984-10-25 |
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